title: An organic acid based counter selection system for cyanobacteria.
abstract: Cyanobacteria are valuable organisms for studying the physiology of photosynthesis and carbon fixation, as well as metabolic engineering for the production of fuels and chemicals. This work describes a novel counter selection method for the cyanobacterium Synechococcus sp. PCC 7002 based on organic acid toxicity. The organic acids acrylate, 3-hydroxypropionate, and propionate were shown to be inhibitory towards Synechococcus sp. PCC 7002 and other cyanobacteria at low concentrations. Inhibition was overcome by a loss of function mutation in the gene acsA, which is annotated as an acetyl-CoA ligase. Loss of AcsA function was used as a basis for an acrylate counter selection method. DNA fragments of interest were inserted into the acsA locus and strains harboring the insertion were isolated on selective medium containing acrylate. This methodology was also used to introduce DNA fragments into a pseudogene, glpK. Application of this method will allow for more advanced genetics and engineering studies in Synechococcus sp. PCC 7002 including the construction of markerless gene deletions and insertions. The acrylate counter-selection could be applied to other cyanobacterial species where AcsA activity confers acrylate sensitivity (e.g. Synechocystis sp. PCC 6803).
authors.affiliation: Microbiology Doctoral Training Program, University of Wisconsin-Madison, Madison, Wisconsin, United States of America ; Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: Matthew B
authors.initials: MB
authors.lastname: Begemann
authors.forename: Erin K
authors.initials: EK
authors.lastname: Zess
authors.forename: Eric M
authors.initials: EM
authors.lastname: Walters
authors.forename: Emily F
authors.initials: EF
authors.lastname: Schmitt
authors.forename: Andrew L
authors.initials: AL
authors.lastname: Markley
authors.forename: Brian F
authors.initials: BF
authors.lastname: Pfleger
mesh_terms: D000179:Acrylates / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D003066:Coenzyme A Ligases / Q000235:genetics* / Q000378:metabolism; D024881:Drug Resistance, Bacterial / Q000187:drug effects / Q000235:genetics*; D019344:Lactic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D060847:Metabolic Engineering / Q000379:methods; D008826:Microbial Sensitivity Tests; D016254:Mutagenesis, Insertional; D009154:Mutation; D011422:Propionates / Q000494:pharmacology; D011544:Pseudogenes; D012641:Selection, Genetic; D046940:Synechococcus / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development
chemical_list: D000179:Acrylates; D001426:Bacterial Proteins; D011422:Propionates; D019344:Lactic Acid; C031601:hydracrylic acid; D003066:Coenzyme A Ligases; C036658:acrylic acid; C029658:propionic acid